EP2 and EP4 agonists as agents for the treatment of influenza a viral infection

ABSTRACT

The present invention is directed to the use of EP2 and/or EP4 agonists as therapeutics for the treatment of diseases associated with influenza A viruses, such as for example H5N1 and mutations thereof.

This application claims the benefit of and priority to U.S. Ser. No.60/859,590, filed on Nov. 16, 2006, which application is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to the use of EP2 and/or EP4 agonistsas therapeutics for the treatment of human respiratory diseasesassociated with influenza A viruses, such as for example H5N1 and itsmutations.

BACKGROUND OF THE INVENTION

The effects of prostaglandins are mediated by their G protein-coupledreceptors which are located on the cell surface. Prostaglandin E₂ (PGE₂)is of particular interest, having a wide variety of cellular effectsthrough binding to functionally different receptor subtypes, namely theEP1, EP2, EP3 and EP4 receptors, all of which respond to PGE₂ but differin their actions.

Dendritic cells (DC) are the most potent antigen-presenting cells of theimmune system. Cytokine production by mature antigen-carrying DC withinlymph nodes is strongly influenced by PGE₂ during their activation inperipheral tissues. Inflammatory cytokines such as IL-1β and TNF-αactivate antigen-carrying DC to secrete IL-12 and promote thedevelopment of T-helper type 1 (Th-1) cytokine expression-biased cells.In contrast, DC activated in the presence of PGE₂ show impaired IL-12production and promote the development of T-helper type 2 (Th-2)cytokine expression-biased cells [Hilkens C M et al., J. Immunol.156:1722-27 (1996)]. The difference in the ability to produce IL-12 inresponse to PGE₂, established during DC activation in the peripheraltissues, is stable to the removal of cytokines and PGE₂.

Increased production of cytokines triggers inflammation, a normalresponse by the body to help fight a virus. However, when cytokineproduction becomes prolonged or excessive it can inflame airways, makingit hard to breathe, which in turn can result in pneumonia and acuterespiratory distress; and it can injure other organs, which can resultin severe life-threatening complications.

It has recently been demonstrated that influenza A subtype H5N1 virusesassociated with the recent outbreaks of avian flu in Asia are morepotent inducers of inflammatory cytokines and chemokines in primaryhuman alveolar and bronchial epithelial cells in vitro in comparison tothe more common, less virulent human flu virus H1N1. Levels of cytokinesand chemokines were from 3 times to more than 10 times higher in thehuman cells infected with the H5N1 virus than those infected with H1N1(N C W Chan, et al. Respiratory Research 2005, 6:135; article URL:http://respiratory-research.com/content/6/1/135).

These test data correlate with the high levels of cytokines andchemokines seen in patients afflicted with the avian flu, indicatingthat the hyper-induction of cytokines and/or chemokines is likelyrelevant to the pathogenesis of human H5N1 disease. Standard steroidanti-inflammatory therapy against avian flu has been of littletherapeutic value. Tamiflu has shown efficacy in that mice infected withH5N1 influenza virus survived when treated. For cases of human infectionwith H5N1, Tamiflu may improve prospects for survival but clinical dataare limited. Concerns have been recently raised about the safety ofTamiflu treatment to patients having the avian flu.

It would therefore be desirable to have a therapeutic agent thatinhibits the release of overstimulated cytokines and chemokines,especially TNFα interferon gamma (IFN-γ) and Interferon gamma. It wouldalso be desirable to have a therapeutic agent that would treat diseasesassociated with human H5N1 and other influenza A subtype viruses whilebeing well-tolerated by the patients.

European patent EP 1306087 describes EP2 receptor agonists which areused in the treatment of erectile dysfunction. The same structural classis described in European patent EP 860430, and their use for producing amedicament for the treatment of immunological disorders, asthma andabortion is claimed. PCT publication WO 04/32965 describes EP2 receptoragonists which are used for the treatment and prevention of disorderscaused by an organ dysfunction caused by ischemia. WO 04/009117describes EP2 and EP4 receptor agonists for the treatment of disorderscaused by uterine contraction, for example painful menstruation. WO03/074483 and WO 03/009872 describe agonists which bind equally to theEP2 and the EP4 receptor (Ono Pharmaceuticals). Agonists of the EP2 andof the EP4 receptor are frequently described in connection with thetreatment of osteoporosis (WO 99/19300, US 2003/0166631, WO 03/77910, WO03/45371, WO 03/74483 and WO 03/09872) and for glaucoma treatment (WO04/37813, WO 04/37786, WO 04/19938, WO 03/103772, WO 03/103664, U.S.Pat. No. 6,747,037, U.S. Pat. No. 6,410,591, WO 03/40123, WO 03/47513,WO 03/47417). WO 04/12656 claims EP2 receptor agonists in connectionwith inflammation. WO 03/77919 claims EP4 receptor agonists for thetreatment of fertility.

SUMMARY OF THE INVENTION

The present invention is directed to agents that are useful astherapeutics against viral diseases. More particularly, the invention isdirected to a method of treating diseases associated with influenza Avirus, and especially with the influenza A subtype H5N1 virus. Themethod comprises administering to a patient in need thereof an effectiveamount of an EP2 agonist, an EP4 agonist, a mixed EP2/EP4 agonist, ormixtures thereof (all of which are encompassed herein under the terms“EP2 and EP4 agonists”, “EP2 and/or EP4 agonists”, and “EP agonists”).

DETAILED DESCRIPTION OF THE INVENTION

EP2, EP4, and mixed EP2/EP4 agonists useful in the present inventioninclude all those that inhibit the release of cytokines and/orchemokines in response to infection by influenza A viruses and, in apreferably preferred embodiment, by the influenza A subtype H5N1 virus.Such inhibition can be determined by one of skill in the art by methodsknown in the art or as taught herein, without undue experimentation.

In one presently preferred embodiment, the agonists are selected from5-cyanoprostacyclin derivatives. 5-Cyanoprostacyclin derivatives andcertain of their pharmacological effects are known from U.S. Pat. Nos.4,219,479 and 5,049,582, the entire disclosures of which areincorporated herein by reference. It is believed that these compoundsexhibit both EP2 and EP4 agonistic activity (a mixed EP2/EP4 agonist).The production of these compounds and the pharmaceutically acceptablesalts thereof are described in detail in the above US patents.Cyclodextrin clathrates of the 5-cyano-prostacyclin derivatives are alsoincluded within the scope of the present invention; they are disclosedand claimed in U.S. Pat. No. 5,010,065, the entire disclosure of whichis incorporated herein by reference. The above 5-cyanoprostacyclinderivatives have not been previously disclosed as being effective in thetreatment or prevention of viral diseases, and this new pharmacologicalproperty also has no direct connection with the effects described in theUS patents.

In a presently preferred embodiment, the 5-cyanoprostacyclin derivativeuseful in treating viral diseases according to the present invention isNileprost, 5-cyano-15-methylprostacyclin:

It has now been found that the above-described 5-cyanoprostacyclinderivatives inhibit the release of Th-1 cytokines while sparing theexpression of Th-2 cytokines and enhance a polarization of T cellsrecruitment towards the Th-2 response and away from the Th-1 response.This makes them desirable as pharmaceuticals for treating diseasesassociated with viruses and particularly with the influenza A H5N1subtype. This is particularly true since they are distinguished overnatural prostaglandins by an improved specificity, longer period ofeffectiveness and higher stability. Additionally, these compounds havebeen found in clinical phase I studies to be well-tolerated by humansand to have no hypotensive effects, making them further suitable aspharmaceuticals and anti-flu therapeutics.

In a second presently preferred embodiment of the invention, the EP2agonist is selected from certain prostacyclin and carbacyclinderivatives that are disclosed, together with methods for theirsynthesis, in, e.g., U.S. Pat. Nos. 4,423,067, 4,474,802, 4,692,464,4,708,963, 5,013,758 and/or CA 1248525, the entire disclosures of whichare incorporated herein by reference. These prostacyclin and carbacyclinderivatives have not been previously disclosed as being effective in thetreatment or prevention of viral diseases, and this new pharmacologicalproperty also has no direct connection with the effects described in theUS patents.

In a presently preferred embodiment, the prostacyclin or carbacyclinderivative useful in treating viral diseases according to the presentinvention is selected from Iloprost, Cicaprost, Eptaloprost, Beraprost,and Ciprosten. In a more preferred embodiment, the prostacyclin analogis Beraprost,(+/−)-(1R*,2R*,3as*,8bS*)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butyrate:

In yet another embodiment of the invention, the EP2 agonist is selectedfrom the 9-chloro-15-deoxyprostaglandin derivatives of the generalformula I:

wherein

-   R¹ is a CH₂OH, a —COOR², a —CONHR² or a —CONHR³ group,-   R² is a hydrogen,-    a C₁-C₁₀-alkyl radical which is linear or branched, optionally    mono- to polyunsaturated and is optionally mono- to poly-substituted    by halogen, C₁-C₄-alkoxy, substituted C₃-C₁₀-aryl, optionally    substituted C₃-C₁₀-aroyl, optionally substituted di-C₁-C₅-alkylamino    or optionally substituted tri-C₁-C₅-alkylamino,-    a C₃-C₁₀-cycloalkyl which is optionally substituted by C₁-C₄-alkyl,-    a C₃-C₁₀-aryl which is optionally substituted by phenyl,    1-naphthyl, 2-naphthyl which in turn may be substituted in position    3 and in position 4 by fluorine, chlorine, alkoxy or trifluoromethyl    or in position 4 by hydroxy, halogen, phenyl, one or more    C₁-C₄-alkyl groups, chloromethyl, fluoromethyl, trifluoromethyl,    carboxy, hydroxy or C₁-C₄-alkoxy,-    or a C₃-C₇-heterocycloalkyl,-   R³ is a C₁-C₁₅-carboxylic acid or a C₁-C₁₅-sulphonic acid,-   A is a cis-CH═CH— or —CH₂—CH₂— group,-   B is a trans-CH═CH— or —CH₂—CH₂— group,-   W is a C₂-C₆-alkylene,-   R⁴ is a hydroxy group, a radical O—R⁶ or O—R⁷, where R⁶ is a    tetrahydropyranyl, tetrahydrofuranyl, trimethylsilyl,    tert-butyldimethylsilyl, tert-butyldiphenylsilyl or tribenzylsilyl    radical and R⁷ is a C₁-C₁₅-carboxylic acid,-   R⁵ is a hydrogen, a C₁-C₁₀-alkyl or a C₁-C₁₀-alkenyl group,-   n is the number 1-4,    and the salts thereof and the cyclodextrin clathrates thereof with    physiologically tolerated bases.

Alkyl groups are linear or branched alkyl groups, saturated andunsaturated alkyl radicals having 1-10 C atoms. Examples which may bementioned are methyl, ethyl, propyl, butyl, isobutyl, tert-butyl,pentyl, neopentyl, hexyl, heptyl, octyl, decyl, butenyl, isobutenyl,propenyl, pentenyl, benzyl, m- and p-chlorobenzyl groups. The alkylgroups may optionally be mono- to polysubstituted by halogen atoms (e.g.fluorine, chlorine or bromine), by alkoxy groups (such as, for example,methoxy, ethoxy or propoxy), by substituted aryl or aroyl groups (e.g.phenyl), or by dialkylamino (e.g. dimethylamino, diethylamino,dimethylaminopropyl or trialkylammonium), where monosubstitution is tobe preferred.

Suitable aryl groups are both substituted and unsubstituted aryl groupssuch as, but not limited to, phenyl, 1-naphthyl and 2-naphthyl.Substituents may be selected from, for example, 1 to 3 halogen atoms, aphenyl group, 1 to 3 alkyl groups each having 1-4 carbon atoms,chloromethyl, fluoromethyl, trifluoromethyl, carboxy, hydroxyl, or analkoxy group having 1-4 carbon atoms.

The cycloalkyl group comprises 3-10 carbon atoms in the ring. The ringmay be substituted by alkyl groups having 1-4 carbon atoms. Examples ofcycloalkyl groups include, but are not limited to, cyclopentyl,cyclohexyl, methylcyclohexyl and adamantyl.

Suitable heterocyclic groups are 5- and 6-membered heterocycles whichcomprise at least 1 heteroatom, preferably nitrogen, oxygen or sulphur.Examples include, but are not limited to, 2-furyl, 2-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, oxazolyl, thiazolyl, pyrimidinyl, pyridazinyl,pyrazinyl, 3-furyl, 3-thienyl, and 2-tetrazolyl.

Physiologically tolerated acid residues are suitable as acid residue.Preferred acids are organic carboxylic acids and sulphonic acids having1-15 carbon atoms which belong to the aliphatic, cycloaliphatic,aromatic, and heterocyclic series. Examples which may be mentioned ofsubstituents are C₁-C₁₅-alkyl, hydroxy, C₁-C₁₅-alkoxy, oxo groups, aminogroups and halogen atoms. Examples of carboxylic acids which may bementioned are formic acid, acetic acid, propionic acid, butyric acid,isobutyric acid, valeric acid, isovaleric acid, caproic acid, oenanthicacid, caprylic acid, pelargonic acid, capric acid, undecylic acid,lauric acid, tridecylic acid, myristic acid, pentadecylic acid,trimethylacetic acid, diethylacetic acid, tert-butylacetic acid,cyclopropylacetic acid, cyclopentylacetic acid, cyclohexylacetic acid,cyclopropanecarboxylic acid, cyclohexanecarboxylic acid, phenylaceticacid, phenoxyacetic acid, methoxyacetic acid, ethoxyacetic acid, mono-,di- and trichloroacetic acid, aminoacetic acid, diethylaminoacetic acid,piperidinoacetic acid, morpholinoacetic acid, lactic acid, succinicacid, adipic acid, benzoic acid, benzoic acids substituted by halogen,trifluoromethyl, hydroxy, alkoxy or carboxy groups, nicotinic acid,isonicotinic acid, furan-2-carboxylic acid, and cyclopentylpropionicacid. Examples of suitable sulphonic acids are methanesulphonic acid,ethanesulphonic acid, isopropanesulphonic acid, β-chloroethanesulphonicacid, butanesulphonic acid, cyclopentanesulphonic acid,cyclohexanesulphonic acid, benzenesulphonic acid, p-toluenesulphonicacid, p-chlorobenzenesulphonic acid, N,N-dimethylaminosulphonic acid,N,N-diethylaminosulphonic acid, N,N-bis(β-chloroethyl)aminosulphonicacid, N,N-diisobutylaminosulphonic acid, N,N-dibutylaminosulphonic acid,pyrrolidino-, piperidino-, piperazino-, N-methylpiperazino- andmorpholinosulphonic acid.

The hydroxy group may be functionally modified, for example byetherification or esterification.

Suitable ether residues are the residues known to the skilled person.Preference is given to ether residues which can easily be eliminated,such as, for example, the tetrahydropyranyl, tetrahydrofuranyl,trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl ortribenzylsilyl radicals.

Suitable acyl radicals are the carboxylic acids mentioned under R⁷.Examples of those which may be mentioned by name are acetyl, propionyl,butyryl and benzoyl.

Suitable for the salt formation are inorganic and organic bases as knownto the skilled person for the formation of physiologically toleratedsalts. Examples which may be mentioned are alkali metal hydroxides suchas sodium and potassium hydroxides, alkaline earth metal hydroxides,such as calcium hydroxide, ammonia, amines such as ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,tris(hydroxymethyl)methylamine, etc.

Compounds of the general formula I which have proved to be particularlyeffective are those where

-   R¹ is a CH₂OH, —COOR², —CONHR² or —CONHR³ group,-   R² is a hydrogen or-    a C₁-C₁₀-alkyl radical which is linear or branched, optionally    mono- to polyunsaturated and is optionally monosubstituted by    fluorine, chlorine or bromine, by C₁-C₄-alkoxy, substituted    C₃-C₁₀-aryl or optionally substituted C₃-C₁₀-aroyl,    di-C₁-C₅-alkylamino or tri-C₁-C₅-alkylamino,-    a C₅-C₆-cycloalkyl which is optionally substituted by C₁-C₄-alkyl,-    a C₃-C₁₀-aryl radical which is optionally substituted by phenyl    which may be substituted in position 3 or 4 by fluorine, chlorine,    alkoxy or trifluoromethyl or in position 4 by hydroxy,-    a C₅-C₆-heterocycloalkyl which may be interrupted one or more times    by nitrogen, oxygen or sulphur,-   R³ is a C₁-C₁₀-carboxylic acid or C₁-C₁₀-sulphonic acid,-   A is a cis-CH═CH— or —CH₂—CH₂— group,-   B is a trans-CH═CH— or —CH₂—CH₂— group,-   W is a C₂-C₆-alkylene,-   R⁴ is a hydroxy group,-   R⁵ is a hydrogen, a C₁-C₆-alkyl or C₁-C₁₀-alkenyl group and-   n is the number 1-4, preferably 2-3.

Compounds of the general formula I which have proved to be veryparticularly effective are those where

-   R¹ is a —CH₂OH, —COOR²—CONHR² or —CONHR³ group,-   R² is a hydrogen or a C₁-C₄-alkyl which is optionally substituted by    phenyl,-    a C₅-C₆-cycloalkyl,-    a C₃-C₆-aryl which is optionally substituted by phenyl,-   R³ is a C₁-C₆-carboxylic acid or C₁-C₆-sulphonic acid,-   A is a cis-CH═CH— or —CH₂—CH₂— group,-   B is a trans-CH═CH— or —CH₂—CH₂— group,-   W is a C₂-alkylene,-   R⁴ is a hydroxy group,-   R⁵ is a hydrogen, saturated C₁-C₄-alkyl or C₁-C₅-alkenyl, and-   n is the number 1-4, preferably 2-3, more preferably 2.

In a presently preferred embodiment, the EP2 agonist useful in treatingautoimmune diseases according to the present invention is the9-chloro-15-deoxyprostaglandin derivative(5Z,13E)-(9R,11R)-9-chloro-11-hydroxy-17,17-tetramethylene-20-nor-5,13-prostadienoicacid.

The prostane derivatives of the above general formula I are prepared bythe reaction of an aldehyde of the general formula II

(wherein R¹ has the meaning of the radicals —COOR², —CONHR³, and A, andR⁴ is as defined above, where the free OH group in R⁴ is protected)with the carbanion of the sulphone of the general formula III

Acetylation of the resulting hydroxysulphone is followed by reductiveelimination to give the olefin and, where appropriate, a subsequentdeprotection of the hydroxy groups which are protected in any sequenceand, where appropriate, esterification, etherification and/orhydrogenation of double bonds and/or esterification of an esterifiedcarboxy group (R¹═COOR²) and/or of a free carboxy group (COOR² withR²═H) and/or conversion of a free carboxy group (COOR² with R²═H) intoan amide (R¹═CONR²) and/or reduction of a free or esterified carboxygroup (R¹═CONHR³).

Reaction of the aldehyde of the general formula II with the carbaniongenerated from the sulphone III takes place in a manner known per seusing an inert solvent such as, for example, tetrahydrofuran or diethylether at temperatures between −100° C. and 24° C., preferably −100° C.to −70° C. The carbanion of the sulphone III is generated in aconventional way with a base such as, for example, butyllithium,methyllithium, potassium tert-butoxide, sodium hydride, lithiumdiisopropylamide, preferably butyllithium. The carbanion formation iscarried out at temperatures from −78° C. to 25° C., preferably at −78°C.

Acetylation of the generated hydroxy group takes place in a known mannerwith acetic anhydride, where appropriate in the presence of a base, forexample pyridine, at temperatures between −78° C. and 25° C.

Reductive elimination of the intermediate acetoxy sulphone to give thetrans-olefin of the general formula I takes place with magnesium powderin methanol with the addition of a catalytic amount ofchlorotrimethylsilane. The reaction is carried out at temperaturesbetween 0° C. and 60° C., preferably between 15° C. and 25° C.Alternatively, the reductive elimination can also be carried out withsodium amalgam.

Reduction to give the compounds of the general formula I with R¹ in themeaning of a —CH₂OH group is carried out with a reducing agent suitablefor reducing esters or carboxylic acids, such as, for example, lithiumaluminium hydride, diisobutylaluminium hydride etc. Suitable solventsare diethyl ether, tetrahydrofuran, dimethoxyethane, toluene etc. Thereduction is carried out at temperatures from −30° C. to the boilingpoint of the solvent used, preferably 0° C. to 30° C.

Functionally modified hydroxy groups are liberated by known methods. Forexample, elimination of hydroxy protective groups such as, for example,the tetrahydropyranyl radical is carried out in an aqueous solution ofan organic acid, such as, for example, oxalic acid, acetic acid,propionic acid, inter alia, or in an aqueous solution of an inorganicacid such as, for example, hydrochloric acid. It is expedient to add awater-miscible inert organic solvent to improve the solubility. Examplesof suitable organic solvents are alcohols such as methanol and ethanol,and ethers such as dimethoxyethane, dioxane and tetrahydrofuran.Tetrahydrofuran is preferably used. The elimination is preferablycarried out at temperatures between 20° C. and 80° C.

The acyl groups are hydrolysed for example with alkali metal or alkalineearth metal carbonates or hydroxides in an alcohol or in the aqueoussolution of an alcohol. Suitable alcohols are aliphatic alcohols suchas, for example, methanol, ethanol, butanol etc., preferably methanol.Alkali metal carbonates and hydroxides which may be mentioned arepotassium and sodium salts. The potassium salts are preferred.

Examples of suitable alkaline earth carbonates and hydroxides arecalcium carbonate, calcium hydroxide and barium carbonate. The reactiontakes place at from −10° C. to +70° C., preferably at +25° C.

The ester group —COOR² for R¹, for example in which R² is an alkyl grouphaving 1-10 C atoms, is introduced by the methods known to the skilledperson. The 1-carboxy compounds are reacted for example withdiazohydrocarbons in a manner known per se. Esterification withdiazohydrocarbons takes place for example by mixing a solution of thediazohydrocarbon in an inert solvent, preferably in diethyl ether, withthe 1-carboxy compound in the same or in a different inert solvent, suchas, for example, methylene chloride. After the reaction is complete in 1to 30 minutes, the solvent is removed and the ester is purified in aconventional way. Diazoalkanes are either known or can be prepared byknown methods (Org. Reactions Vol. 8, pages 389-394 (1954)).

The ester group COOR² for R¹, in which R² is a substituted orunsubstituted aryl group, is introduced by the methods known to theskilled person. For example, the 1-carboxy compounds are reacted withthe appropriate aryl hydroxy compounds with dicyclohexylcarbodiimide inthe presence of a suitable base, for example pyridine, DMAP,triethylamine, in an inert solvent. Suitable solvents are methylenechloride, ethylene chloride, chloroform, ethyl acetate, tetrahydrofuran,preferably chloroform. The reaction is carried out at temperaturesbetween −30° C. and +50° C., preferably at 10° C.

If it is intended to reduce C═C double bonds present in the initialproduct, the hydrogenation takes place by methods known per se.

Hydrogenation of the 5,6 double bond is carried out in a manner knownper se at low temperatures, preferably at about −20° C., in a hydrogenatmosphere in the presence of a noble metal catalyst. An example of asuitable catalyst is 10% palladium on carbon.

If both the 5,6 double bond and the 13,14 double bond are hydrogenated,a higher temperature is used, preferably about 20° C.

The prostaglandin derivatives of the general formula I with R² meaning ahydrogen atom can be converted into a salt by neutralization usingsuitable amounts of the appropriate inorganic bases. For example,dissolving the appropriate prostaglandin acids in water containing thestoichiometric amount of the base results, after the water has beenevaporated off or a water-miscible solvent, e.g. alcohol or acetone, hasbeen added, in the solid organic salt.

An amine salt is prepared in a conventional way by dissolving theprostaglandin acid for example in a suitable solvent, for exampleethanol, acetone, diethyl ether, acetonitrile or benzene, and adding atleast the stoichiometric amount of the amine to this solution. Thisusually results in the salt in solid form, or it is isolated in theusual way after evaporation of the solvent.

The amide group —CONHR³ for R¹ is introduced by methods known to theskilled person. The carboxylic acids of the general formula I (R²═H) areinitially converted into the mixed anhydride with isobutyl chloroformatein the presence of a tertiary amine such as, for example, triethylamine.Reaction of the mixed anhydride with the alkali metal salt of theappropriate amine or with ammonia (R³═H) takes place in an inert solventor solvent mixture, such as, for example, tetrahydrofuran,dimethoxyethane, dimethylformamide, hexamethylphosphoric triamide, attemperatures between −30° C. and +60° C., preferably at 0° C. to 30° C.

A further possibility for introducing the amide group —CONHR³ for R¹consists in reacting a 1-carboxylic acid of the general formula I(R²═H), in which there is optionally intermediate protection of freehydroxy groups, with compounds of the general formula IVO═C═N—R³  IVin which R³ has the meaning indicated above.

Reaction of the compound of the general formula I (R²═H) with anisocyanate of the general formula IV takes place where appropriate withaddition of a tertiary amine such as, for example, triethylamine orpyridine. The reaction can be carried out without solvent or in an inertsolvent, preferably acetonitrile, tetrahydrofuran, acetone,dimethylacetamide, methylene chloride, diethyl ether, toluene, attemperatures between −80° C. to 100° C., preferably at 0° C. to 30° C.

If the starting material comprises OH groups in the prostane residue,these OH groups are also reacted. If the final products eventuallydesired comprise free hydroxy groups in the prostane residue, it isexpedient to start from starting materials with intermediate protectionthereof by ether or acyl radicals which can preferably be easilyeliminated.

The aldehydes of the general formula II which are used as startingmaterial are known or can be prepared for example by selectiveepoxidation in a manner known per se of the 13,14 double bond of a9-haloprostaglandin of the general formula V, preferably with R¹ meaninga —COOCH₃ group, with tert-butyl hydroperoxide and titanium(IV)isopropoxide in methylene chloride at −20° C.

Subsequent epoxide cleavage with periodic acid in diethyl ether and,where appropriate, protection of the 11-hydroxy group, for example withdihydropyran, affords the aldehyde of the general formula II.

The sulphone of the general formula III used as starting material can beprepared from cycloalkylcarboxylic acids of the general formula VII inwhich n has the meaning indicated above by alkylation with an alkylhalide of the general formula VIII in which R⁵ has the meaning indicatedabove, and halogen can be iodine, chlorine or bromine.

Esterification of IX with methyl iodide and potassium carbonate inacetone is followed by reduction of the resulting methyl ester to thealcohol with lithium aluminium hydride in diethyl ether. Oxidation ofthe alcohol with SO₂-pyridine complex in the presence of triethylaminein a mixture of dimethyl sulphoxide and methylene chloride affords thealdehyde of the general formula X. Subsequent Wittig-Horner reactionand, where appropriate, hydrogenation of the double bond to give XIleads, after reduction with diisobutylaluminium hydride, to the alcoholof the general formula XII. Hydrogenation of the double bond can,however, also be carried out after reduction of the ester XI to thealcohol XII, with W meaning a double bond.

Subsequent replacement of the hydroxy group takes place afterintermediate tosylation by reaction with thiophenol in toluene. Thethioether XIII obtained in this way is finally oxidized in an aqueousmethanolic solution to the sulphone of the general formula III.

In a further embodiment of the invention, the EP2 and EP4 agonists areselected from those reported by Ono Pharmaceuticals, by Merck and byPfizer.

The pharmacologically active EP2 and EP4 agonists useful in the presentinvention can be processed in accordance with conventional methods ofgalenic pharmacy to produce medicinal agents for treating diseasesassociated with influenza A viruses, and particularly the H5N1 virus.The pharmaceutical compositions comprise the EP agonist in an effectiveamount (that is, an amount effective to treat an influenza A viraldisease) and one or more pharmaceutically acceptable excipients.

Suitable excipients may include, but are not limited to, pharmaceutical,organic or inorganic inert carrier materials suitable for enteral,parenteral or topical administration which do not deleteriously reactwith the active compounds. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,gelatine, gum arabic, lactate, starch, magnesium stearate, talc,vegetable oils, polyalkylene glycols, polyvinyl pyrrolidone,hydroxyl-methylcellulose, silicic acid, viscous paraffin, fatty acidmonoglycerides and diglycerides, and the like. The pharmaceuticalproducts may be in solid form, for example as tablets, coated tablets,suppositories or capsules, or in liquid form, for example as solutions,suspensions or emulsions. They may additionally comprise, whereappropriate, auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts to alter the osmoticpressure, buffers, coloring, flavoring, and/or aromatic substances andthe like that do not deleteriously react with the active compounds.Examples of suitable pharmaceutical compositions include the following:

Aerosol solutions are expediently produced for delivery via inhalation.

Particularly suitable for oral use are tablets, coated tablets orcapsules with talc and/or carbohydrate carriers or binders, such as, forexample, lactose, maize starch or potato starch. Use is also possible inliquid form, such as, for example, as fluid to which a sweetener isadded where appropriate.

Sterile, injectable, aqueous or oily solutions are used for parenteraladministration, as well as suspensions, emulsions or implants, includingsuppositories. Ampoules are convenient unit dosages. Sustained releasecompositions can be formulated including those wherein the activecompound is protected with differentially degradable coatings, e.g., bymicroencapsulation, multiple coatings, etc.

Carrier systems which can also be used are surface-active excipientssuch as salts of bile acids or animal or vegetable phospholipids, butalso mixtures thereof, and liposomes or constituents thereof.Transdermal patches may also be used as delivery means.

The dosage of the EP2 and/or EP4 therapeutic agent(s) will be thatamount that is effective to treat an influenza A viral disease. Theeffective amount of active ingredient may vary depending on the route ofadministration, the age and weight of the patient, the nature andseverity of the disorder to be treated, and similar factors. Theeffective amount can be determined by methods known to those of skill inthe art. The daily dose is generally about 0.1-200 μg/kg/day, preferablyabout 0.5-10 μg/kg/day, when administered to human patients, it beingpossible for the dose to be given as a single dose to be administeredonce or divided into two or more daily doses.

The EP2 and/or EP4 agonists may be delivered as a co-treatment togetherwith other anti-viral or anti-inflammatory compounds, such as, but notlimited to, oseltamivir (Tamiflu™) and zanamivir (Relenza™). Thecompounds may be delivered to the patient at the same time orsequentially as separate formulations, or they may be combined anddelivered as a single formulation.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever.

EXAMPLES Example 1 Demonstration of Nileprost Inhibition of T LymphocyteTh-1 Cytokine Release

Principle:

The activation of a human T lymphocyte by an antigen-presenting cell andantigen through the T cell receptor is mimicked in experimentalconditions by the lectin Concanavilin A (ConA). It is known that ConAbinds to the T cell receptor and stimulates the cell to release variouscytokines. The binding of Nileprost to the EP receptor inhibits variouscytokine release. One of the Th-1 released cytokines is IFN-γ. Thebiochemistry and biological activities of IFN-γ have been extensivelyreviewed in the literature.

Detection Method:

Human IFN-γ is a dimer of the expressed 143 amino acid protein.Enzyme-linked immunosorbant assays (ELISA) based on antibodies specificto IFN-γ are commercially available. Standards and samples are pipettedinto the wells of a microplate. An antibody specific to human IFN-γ isadded to the wells. A substrate is added to the wells and color developsin proportion to the amount of IFN-γ bound. The intensity of the coloris measured.

Procedure:

Peripheral blood lymphocytes were isolated from human donors using aFicoll density gradient and residual erythrocytes were removed byselective lysis. The lymphocytes are cultured at approximately 10⁶ cellsper mL in RPMI1640 with 10% additional fetal bovine serum. The cellcultures were activated with 2 μg/ml of ConA as described above.Nileprost was added at various dilutions during the ConA activation.Cells were incubated for approximately 18 hr at 37° C. IFN-γ releasedduring activation was measured by ELISA.

Nileprost Inhibition (percent) human T cells ConA stimulation Percentinhibition of IFN-γ compared to uninhibited control Nileprost (nM) 0 0.51.25 2.5 5 10 20 Donor 1  0% 17% 23% 45% 56% 50% 67% std. deviation 21%21%  3% 11% 10% 10%  5% Donor 2  0%  2% 11% 17% 35% 43% 41% std.deviation 17% 29% 21% 19%  6% 10%  7% Donor 3  0% 26% 36% 43% 54% 64%68% std. deviation 12%  9%  2%  3%  2%  7% 10% Donor 4  0% 21% 29% 31%48% 55% 56% std. deviation 13%  6% 12%  8%  8%  5%  6% Nileprost (nM) 00.04 0.11 1 3 9 83 250 Donor 5 0% 16% 22% 43% 56% 59% 57% 59% Std.deviation 6%  0%  9%  3%  1%  9%  3%  0% Donor 6 0%  7% 16% 32% 46% 60%60% 76% Std. deviation 6% 20% 19% 13% 14% 10%  4%  1% Donor 7 0%  9% 22%24% 22% 48% 65% 57% Std. deviation 11%   6% 11% 11%  5%  8%  3% 15%Donor 8 0% 27% 34% 51% 71% 73% 85% 83% Std. deviation 15%  10%  6%  2% 3%  5%  5%  4% Nileprost showed, dose responsively, very highinhibitory activity in the T lymphocyte Th-1 cytokine release.

Example 2 Demonstration of Nileprost Inhibition of ProinflammatoryCytokine Release

Principle:

The activation of a human T lymphocyte by an antigen-presenting cell andantigen through the T cell receptor is mimicked in experimentalconditions by the lectin Concanavilin A (ConA). It is known that ConAbinds to the T cell receptor and stimulates the cell to release variouscytokines. The binding of Nileprost to the EP receptor inhibits variouscytokine release. One of the pro-inflammatory cytokines inhibited isTNFα. The biochemistry and biological activities of TNFα have beenextensively reviewed in the literature.

Detection Method:

Human TNFα is a trimer of the expressed protein. Enzyme-linkedimmunosorbant assays (ELISA) based on antibodies specific to TNFα arecommercially available. Standards and samples are pipetted into thewells of a microplate. An antibody specific to human TNFα is added tothe wells. A substrate is added to the wells and color develops inproportion to the amount of TNFα bound. The intensity of the color ismeasured.

Procedure:

Peripheral blood lymphocytes were isolated from human donors using aFicoll density gradient and residual erythrocytes were removed byselective lysis. The lymphocytes are cultured at approximately 10⁶ cellsper mL in RPMI1640 with 10% additional fetal bovine serum. The cellcultures were activated with 2 μg/ml of ConA as described above.Nileprost was added at various dilutions during the ConA activation.Cells were incubated for approximately 18 hr at 37° C. TNFα releasedduring activation was measured by ELISA.

Nileprost Inhibition (percent) human T cells ConA stimulation Percentinhibition of TNFα compared to uninhibited control Nileprost (nM) 0 0.51.25 2.5 5 10 20 Donor 1 0%  7% 14% 24% 47% 46% 50% Std. deviation 21% 15%  4% 17% 14%  9% 22% Donor 2 0%  1%  5% 19% 36% 49% 46% Std.deviation 2% 15% 15%  5%  4%  9%  5% Nileprost (nM) 0 0.04 0.11 1 3 9 83250 Donor 3 0%  1%  4% 24% 28% 53% 61% 49% Std. deviation 21% 22% 32%13%  4%  4% 15% 30% Donor 4  0% 11% 16% 37% 48% 67% 76% 76% Std.deviation 17%  2% 14%  9%  5%  5%  3%  6%

Example 3 Demonstration of Nileprost Inhibition of Cytotoxic CD8+Lymphocyte Cytokine Release

Principle:

The activation of a human T lymphocyte by an antigen-presenting cell andantigen through the T cell receptor is mimicked in experimentalconditions by the addition of antibodies to the CD3 subunit of the Tcell receptor and antibodies to the CD28 costimulatory receptor. It isknown that anti-CD3 anti-CD28 binding to T lymphocytes stimulates thecells to release various cytokines. Some of these cytokines are IL-2,IFN-γ and GM-CSF. The biochemistry and biological activities of thesecytokines have been extensively reviewed in the literature. The bindingof Nileprost to the EP receptor inhibits the release of various CD8+cytokines.

Detection Method:

Multi-cytokine immunosorbant assays based on antibodies specific tohuman cytokines are commercially available. Standards and samples arepipetted into sample tubes. A monoclonal antibody specific for acytokine is covalently linked to a fluorescent bead set, which capturesthe cytokine. A complementary biotinylated monoclonal cytokine antibodythen completes the immunological sandwich and the reaction is detectedwith streptavidin-phycoerythrin.

Procedure:

CD14-negative populations were isolated from four donors by MiltenyiCD14 beads. The four populations were allowed to rest at 5×10⁶/mL inseparate flasks overnight in RPMI 1640, 10% fetal bovine serum.Meanwhile, anti-CD3 antibody (OKT3, functional antibody, eBioscience)were bound to 10 cm plates at 5 μg/mL in sodium carbonate binding bufferat 4° C. The next day, the cells were mixed and 1×09 cells were takenfor CD8 isolation using the Milenyi CD8 isolation kit (negativeselection). The resulting 3.9×10⁸ CD8 cells were resuspended in mediumat 4×10⁶/mL and added to the 10 cm CD3-bound plates. Soluble anti-CD28(2-5 μg/mL) and 1 μM Nileprost or vehicle alone were added to the CD8cells. Incubation was continued overnight and cytokines were detected asdescribed above. The results are presented below.

Nileprost Inhibition (percent) Nileprost standard deviation human CD8cells Anti-CD3/CD28 stimulation Percent inhibition of IFN-γ compared touninhibited control Donor 1 79% 1% Donor 2 59% 1% Donor 3 79% 2% Donor 480% 1% human CD8 cells Anti-CD3/CD28 stimulation Percent inhibition ofGM-CSF compared to uninhibited control Donor 1 77% 0% Donor 2 65% 2%Donor 3 87% 1% Donor 4 61% 6% human CD8 cells Anti-CD3/CD28 stimulationPercent inhibition of IL-2 compared to uninhibited control Donor 1 71%0% Donor 2 68% 4% Donor 3 88% 0% Donor 4 70% 5% Nileprost showed veryhigh inhibitory activity in the cytotoxic CD8+ lymphocyte cytokinerelease.

Example 4 Demonstration of Nileprost Inhibition of HumanMonocyte-Derived Dendritic Cell (DC) Cytokine Release

Principle:

Dendritic cells (DCs) are the most potent antigen-presenting cells andplay a central role in immune response. Following stimulation throughthe toll-like receptors TLR, DCs express and release proinflammatorycytokines and chemokines and may induce activation and proliferation ofnaïve T cells. The binding of Nileprost to the EP receptor inhibits TLR4ligand (LPS)-stimulated IL-12 release. Therefore, Nileprost skews theCD4 T cell differentiation to a Th-2 lineage.

Detection Method:

IL-12 is a 75 kDa glycoprotein heterodimer (p70) composed of twogenetically unrelated subunits linked by a disulfide bond. Enzyme-linkedimmunosorbant assays based on antibodies specific to IL-12 p70 arecommercially available. Standards and samples are pipetted into thewells of a microplate. An antibody specific to human IL-12 is added tothe wells. A substrate is added to the wells and color develops inproportion to the amount of IL-12 bound. The intensity of the color ismeasured.

Procedure:

Human monocyte-derived dendritic cells were isolated from human donorsusing a Ficoll density gradient and residual erythrocytes were removedby selective lysis. CD14 MicroBeads were used for separation of humancells based on the expression of the CD14 antigen. The dendritic cellswere cultured at approximately 1.5×10⁶ cells per mL in RPMI1640 withfetal bovine serum, 200 ng/mL GM-CSF (Leukine) and 10 ng/mL IL-4. Thecells grew for a period of 3 days and then the media was changed. 10ng/mL LPS was used to activate the cells. 1 μM of Nileprost and 1 μM ofPGE₂ were added during the LPS stimulation. Cells were incubated forapproximately 18 hr at 37° C. IL-12 released during activation wasmeasured by ELISA. The results are presented below.

Nileprost Inhibition (percent) Human monocyte-derived dendritic cellsLPS stimulation Percent inhibition of IL-12 compared to uninhibitedcontrol Nileprost PGE2 Donor 1 84% 80% Donor 2 51% 33% Donor 3 69% 83%Donor 4 66% 72% Nileprost showed very high inhibitory activity in humanmonocyte-derived dendritic cell (DC) cytokine release.

Example 5

Human donor monocyte-derived dendritic cells were cultured in RPMI-1640containing 10% fetal bovine serum for six days in the presence of 10ng/mL IL4 and 200 ng/mL GM-CSF. The cells were activated with variousactivation stimuli: 10 ng/mL LPS (Sigma), 5 μg/mL Recombinant humanCD-40 ligand (R&D Systems), or 5 μM Human CpG-DNA (HyCult Biotechnology)in the presence of prostaglandin E2 (PGE₂) (1 μM), vehicle control(DMSO) or test substances (1 μM) for 18 hours. The levels of cellculture supernatant TNFα for individual donors was measured bycommercial ELISA kits. The test substances [results with(5Z,13E)-(9R,11R)-9-chloro-[1-hydroxy-17,17-tetramethylene-20-nor-5,13-prostadienoicacid (Test Substance 1) and another EP agonist (Test Substance 2) areshown in the Table below] led to an inhibition in the cytokine levelsmeasured in the culture supernatant as shown in the Table below.

Inhibition of TNFα Release from activated monocytic cells TestSubstance + Donor 4 Donor 6 LPS (TNFα pg/ml) (TNFα pg/ml) Vehicle 3566.910750.1 PGE2 0.0 174.8 Test Substance 1 643.6 753.7 Test Substance 2179.4 676.1 Test Substance + Donor 3 Donor 4 Donor 6 CD40 ligand (TNFαpg/ml) (TNFα pg/ml) (TNFα pg/ml) Vehicle 567.1 546.3 541.8 PGE2 147.987.0 54.2 Test Substance 1 274.3 226.8 142.4 Test Substance 2 209.4211.2 84.4 Test Substance + Donor 1 Donor 3 Donor 6 CpG (TNFα pg/ml)(TNFα pg/ml) (TNFα pg/ml) Vehicle 2155.9 1340.7 467.8 PGE2 104.5 70.0106.2 Test Substance 1 291.3 599.4 115.9 Test Substance 2 546.9 74.884.8

Example 6 Demonstration of Nileprost Inhibition of TNFα Release fromViral RNA-Activated Human Monocyte-Derived Dendritic Cells

The test substance (Nileprost) was used to inhibit the release of TNFαfrom synthetic viral RNA-activated human monocyte-derived dendriticcells. Cells were cultured in vitro as described in Example 4 andExample 5. Cells were activated with 2 μg/mL Poly(I:C), a syntheticderivative of double-strand RNA (a component of the influenza A virus)in the presence or absence of one micromolar test substance and culturedfor 18 hours. The levels of cell culture supernatant TNFα were measuredby commercial ELISA kits. The test substance (Nileprost) caused asignificant decrease in Poly(I:C)-induced TNFα.

Poly IC-stimulated monocyte-derived dendritic cells TNFα percentinhibition from untreated control Donor 1 Donor 2 Donor 3 Donor 4 PGE296% 63% 81% 29% Nileprost 89% 45% 74% 36% Vehicle  0%  0%  0%  0%

Example 7 Demonstration of Beraprost Inhibition of TNFα Release fromViral RNA-Activated Human Peripheral Blood Lymphocytes

Following the procedures in Example 6, peripheral blood lymphocytes wereisolated and cultured, after which they were activated with Poly(I:C)and further cultured. The levels of cell culture supernatant TNFα wasthen measured by ELISA kits. The test substance (Beraprost) caused asignificant decrease in Poly(I:C)-induced TNFα, as shown in the Tablebelow.

Poly IC-stimulated peripheral blood lymphocytes TNFα percent inhibitionfrom untreated control Beraprost (nM) 0 0.04 0.11 1 3 9 83 250 Donor 1 0% 19% 19% 11% 36% 46% 56% 67% Standard 19% 10% 10%  2%  4% 14% 15%  0%deviation Donor 2  0%  4% 26% 28% 11% 35% 56% 59% Standard 23% 36% 18%10% 32% 31% 16%  5% deviation

Example 8 Tolerance in Humans after Oral Administration of Nileprost

Study A. Tolerance and Pharmacokinetics

14 volunteers were given a single oral dose of Nileprost at 2, 4, 8, 16,32, or 65 μg. The following parameters were studied: Blood pressure,heart rate, clinical chemistry, platelet aggregation, and ECG.

No findings but enteropooling in higher doses, indicating good toleranceto Nileprost.

Pharmacokinetics: mean c_(max) after 32 μg: 840 ± 30 pg/mL 2.1 nM 65 μg:929 ± 254 pg/mL 2.3 nMStudy B. Single Administration up to 375 μg

12 volunteers were given a single oral dose of Nileprost at 50, 75, 112,255 or 375 μg. The following parameters were studied: Blood pressure,heart rate, ECG, lung function, clinical chemistry and hematology,platelet aggregation, and stools.

No findings but “painless diarrhea” in higher doses (255 μg and 375 μg),indicating tolerance to Nileprost.

Pharmacokinetics: mean c_(max) after  50 μg: 566 pg/mL 1.4 nM  75 μg:869 pg/mL 2.2 nM 112 μg: 803 pg/mL 2.0 nM 170 μg: 954 pg/mL 2.4 nM 255μg: 1709 pg/mL  4.3 nM 375 μg: 1716 pg/mL  4.3 nM

1. A method of treating an influenza A virus infection in a patient inneed of such treatment, the method comprising administering to thepatient an effective amount of a therapeutic agent selected from thegroup consisting of nileprost and beraprost.
 2. The method of claim 1,wherein said therapeutic agent is beraprost.
 3. The method of claim 1,wherein said therapeutic agent is nileprost.
 4. The method of claim 1,wherein the influenza A virus is H5N1 or a mutation thereof.
 5. Themethod of claim 2, wherein the influenza A virus is H5N1 or a mutationthereof.
 6. The method of claim 3, wherein the influenza A virus is H5N1or a mutation thereof.
 7. The method according to any one of claim 1, 2,3, 5, or 6, wherein said therapeutic agent inhibits the release ofinflammatory cytokines and/or chemokines in human alveolar and bronchialepithelial cells.
 8. The method of claim 2, wherein said therapeuticagent inhibits the release of inflammatory cytokines and/or chemokinesin human alveolar and bronchial epithelial cells.
 9. The method of claim3, wherein said therapeutic agent inhibits the release of inflammatorycytokines and/or chemokines in human alveolar and bronchial epithelialcells.
 10. The method of claim 4, wherein said therapeutic agentinhibits the release of inflammatory cytokines and/or chemokines inhuman alveolar and bronchial epithelial cells.
 11. The method of claim5, wherein said therapeutic agent inhibits the release of inflammatorycytokines and/or chemokines in human alveolar and bronchial epithelialcells.
 12. The method of claim 6, wherein said therapeutic agentinhibits the release of inflammatory cytokines and/or chemokines inhuman alveolar and bronchial epithelial cells.
 13. The method of claim1, wherein said therapeutic agent inhibits the release of inflammatorycytokines and/or chemokines in peripheral blood lymphocytes.
 14. Themethod of claim 2, wherein said therapeutic agent inhibits the releaseof inflammatory cytokines and/or chemokines in peripheral bloodlymphocytes.
 15. The method of claim 3, wherein said therapeutic agentinhibits the release of inflammatory cytokines and/or chemokines inperipheral blood lymphocytes.
 16. The method of claim 4, wherein saidtherapeutic agent inhibits the release of inflammatory cytokines and/orchemokines in peripheral blood lymphocytes.
 17. The method of claim 5,wherein said therapeutic agent inhibits the release of inflammatorycytokines and/or chemokines in peripheral blood lymphocytes.
 18. Themethod of claim 6, wherein said therapeutic agent inhibits the releaseof inflammatory cytokines and/or chemokines in peripheral bloodlymphocytes.